Painful bladder syndrome/Interstitial cystitis (PBS/IC) is a chronic painful condition of the urinary bladder in which there are no proven etiologies and no effective treatments that are able to ameliorate the symptoms, which include urinary frequency, urgency, nocturia and pain. There is a comparable disease in domestic cats termed feline interstitial cystitis (FIC), which demonstrates nearly all of the characteristics and symptoms of human PBS/IC. Cats with this naturally occurring disease, as opposed to acute injury/inflammation in rodent models, may permit more relevant studies of the pathophysiology of PBS/IC in humans. We have identified a number of abnormalities in the urothelium of FIC bladders including alterations in barrier function, proliferation and growth, and a heightened sensitivity/response to both chemical and physical stimuli. These may be a result of changes in intracellular Ca2+ release/sequestration and/or intracellular signaling pathways. There are also abnormalities in FIC bladder afferents including altered morphological/electrical properties, nerve firing and distribution of nerve fibers. Moreover, we also have evidence of long-term alterations in FIC spinal cord glial cell morphology and function in regions of pelvic afferent input suggesting that these cells play a role in the generation of abnormal sensory mechanisms. The persistent change that takes place in these FIC cell types during long-term culture has given us a different perspective suggesting the probability of multiple changes along the sensory pathway. Recent findings support an important role for activation of spinal cord glial cells in both initiation and amplification of persistent pain, as it is known that these cells can enhance and prolong the response of second order spinal sensory neurons to peripheral stimulation. Thus, multiple sites in the sensory pathway may have a similar type of 'primary' defect, or a primary defect in the urothelium may sequentially trigger a secondary defect at more proximal sites. Our goals in this renewal application are to further understand the signaling pathways underlying the persistent changes observed in urothelial (and glial cell) function, as well as signaling mechanisms responsible for various cell-cell interactions and how these mechanisms may be altered in FIC. These goals will be accomplished using a multidisciplinary approach including imaging techniques, molecular biology and measurement of transmitter release. Aim #1 will evaluate intracellular mechanisms regulating urothelial vesicle recycling and transmitter release in PBS/FIC. We hypothesize that the release of mediators/signaling factors from the urothelium in response to mechanical or chemical stimuli contributes to the sensory symptoms in cats diagnosed with FIC. This aim will utilize imaging with membrane-impermeant dyes in order to explore the mechanisms responsible for chemical and mechanical evoked release of mediators from urothelial cells. Aim #2 will evaluate mechanisms underlying changes in umbrella cell morphology, signaling and communication in PBS/FIC. We will use intact bladder sheets and polarized urothelial cultures to measure changes in apical cell properties and cell signaling between urothelial layers. Aim #3 will evaluate morphological/functional properties of spinal cord glial cells in PBS/FIC. We will characterize the chemistry/morphology and function of cultured spinal cord astrocytes as well as glial-neuronal interactions in normal and FIC. Understanding the mechanisms involved in these types of changes may provide important insights for the development of novel targets for the clinical management of PBS/IC.